Talk with the instructor to make sure your answers are correct. You will need to
ID: 78260 • Letter: T
Question
Talk with the instructor to make sure your answers are correct. You will need to discuss ALL of these questions in your lab report. 1. Which plate should contain the most number of cells growing (assume that transformation is not 100% efficient)? Why? 2. Which plate should contain the least number of cells growing? Why? 2. What do the control plates 1, 3, and 4 tell you (what is the purpose for each of these plates)? Give an explanation for each plate. For example, do you expect growth or no grow the on each plate? If you observed the opposite of what you expect, what does this mean? 3. Which plate will have green cells under a UV light? Why? 4. Which plates will have cells that are NOT green under a UV light? Why? 5. The (-DNA) tube does not contain the pGlo DNA, so the cells will not be transformed with the plasmid. Why does it still have to go through the heat shock process the same way as the (+DNA) tube (be specific)? An organism's environment can determine its physical trait (phenotype). a) After transformation is Successful, what factors must be present in order for you to observe the green fluorescence of the transformed cells (other than the pGlo)? Explain what each factor is doing to cause green fluorescence.Explanation / Answer
1. pGLO plasmid contains ampicilin resistant gene. Plate 4 should contain the most number of cells growing. Because it is not containing ampicilin and thus all the nontransformrd bacteria i.e, -pGLO E.coli will grow on agar. Comparatively less number of cells will grow on Plate 1 and 2 which contain ampicilin because only transformed cells i.e, +pGLO E.coli which are ampicilin resistant will grow and and those are not transformed will not grow (100% transformation is not happened). Plate 3 contains ampicilin and thats why nontransformed cells will not grow which are not ampicilin resistant.
2. Plate 3 should contain least number of cells. Because Plate 3 contains ampicilin and thats why nontransformed cells (-pGLO E.coli) will not grow as it is not ampicilin resistant.
3. Plate 1 tells the number of transformed colonies from which transformation efficiency can be calculated by comparing with plate 4 and selection of transformed cells from nontransformed cells by allowing the growth of only transformed cells on LB/Amp plate. It should show less growth than plate 4 and more growth than olate 3. If the growth we get on plate 1 is more or same the growth on plate 3 then the strain used may be resistant to ampicillin.
Plate 3 should not show any growth as it cnotains ampicilin and cells are nontransformed i.e, not resistant to ampicilin. If still we find growth it indicates that either the nontransformed strain used is resistant to ampicilin or it is contamination of transformed cells or ampicilin resistant bacteria.
Plate 4 tells the total number of nontransformed cells/colonies. It should contain the most number of cells growing because it is not containing ampicilin and thus all the nontransformrd bacteria i.e, -pGLO E.coli will grow on agar. If we get less growth on plate 4 than plate 1 then it may be because of either cells plated were in death phase or very low volume of cell suspension is plated on plate 4(handling error).
4.Plate 2 will have green cells under UV light because it contains arabinose which is neccessary for the expression of GFP gene of the transformed cells. pGLO plasmid has been constructed in which some of the genes needed for digesting arabinose have been replaced by a gene that codes for green fluorescent protein. Because pGLO plasmid contains bidirectional promoter with a gene for metabolizing arabinose, the GFP gene is expressed in the presence of arabinose, which makes the transformed cells express its fluorescence under UV light.
5. plate 1, plate 3, plate 4 will not glow under UV because plate 3 and 4 do not contain transformed (+pGLO cells which contains GFP gene) E.coli. Plate 1 does not contain arabinose hence will not glow thogh it contains transformed cells because a plasmid has been constructed in which some of the genes needed for digesting arabinose have been replaced by a gene that codes for green fluorescent protein. Because it contains a bidirectional promoter with a gene for metabolizing arabinose, the GFP gene is expressed in the presence of arabinose The protein glows green when viewed under UV light.
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